Margalit Maya, Shibolet Oren, Klein Athalia, Elinav Eran, Alper Ruslana, Thalenfeld Barbara, Engelhardt Dean, Rabbani Elazar, Ilan Yaron
Liver Unit, Department of Medicine, Hebrew University-Hadassah Medical Center, Jerusalem, Israel.
Int J Cancer. 2005 Jun 20;115(3):443-9. doi: 10.1002/ijc.20889.
NKT cells are a regulatory subset of T lymphocytes with immune modulatory effects and an important role in anti-tumor immunity. The feasibility of "ex-vivo education" of NKT cells has recently been demonstrated. To evaluate the anti-tumor effect of ex-vivo immune-modulated NKT lymphocytes in a murine model of hepatocellular carcinoma. Athymic Balb/C mice were sublethally irradiated and transplanted with human Hep3B HCC. NKT cells prepared from immunocompetent Balb/C mice were pulsed ex vivo with HCC-derived antigens (Group A), Hep3B cells (group B) or BSA (group C), and adoptively transferred into HCC harboring mice (1 x 0(6) NKT cells per mouse). Group D mice did not undergo NKT cell transplantation. Group E mice were transplanted with 1 x 10(6) NKT cells from HBV-immunized donors. Mice were followed for tumor size and weight. To determine the mechanism of the anti-tumor effect, intrasplenic lymphocyte populations were analyzed by FACS for NKT, CD4+ and CD8+ lymphocyte subpopulations; STAT 1, 4 and 6 expression in splenocytes was assessed by Western blot, and serum cytokine levels were measured by ELISA. Adoptive transfer of NKT cells pulsed with HCC-derived antigens (group A) and NKT cells from immunized donors (group E) resulted in complete disappearance of tumors within 4 weeks and attenuated weight loss (6.5% and 7% in groups A and E, respectively). In contrast, mice in groups B, C, and D developed large, necrotic tumors and severe weight loss (21%, 17% and 23% weight loss in groups B, C, and D, respectively). NKT/CD4 and CD8/CD4 ratios were significantly increased in groups A and E (12.3 and 17.6 in groups A and D, respectively, compared to 6.4, 4.8 and 5.6 in groups B, C and D, respectively, for the NKT/CD4 ratio; 41 and 19.8 in groups A and E, respectively, compared to 6.5, 11.8 and 3.2 in groups B, C, and D, respectively, for the CD8/CD4 ratio). Expression of the transcription factor STAT4 was evident in group A, but not in groups B-D. Serum IFNgamma, IL12 and IL4 levels were increased in groups A and E. Adoptive transfer of NKT lymphocytes exposed ex vivo by HCC-derived antigens loaded on dendritic cells and NKT cells from immunized donors led to suppression of HCC in mice. NKT-mediated anti-tumor activity was associated increased NKT and CD8+ T lymphocyte numbers, increased expression of STAT4, a marker for IL-12 activity and elevated serum levels of the proinflammatory cytokines IFNgamma and IL12, and of IL4. Ex-vivo modulation of NKT lymphocytes holds promise as a novel mode of immune therapy for HCC.
自然杀伤T细胞（NKT细胞）是T淋巴细胞的一个调节亚群，具有免疫调节作用，在抗肿瘤免疫中发挥重要作用。最近已证明了NKT细胞“体外培养”的可行性。为了评估体外免疫调节的NKT淋巴细胞在小鼠肝细胞癌模型中的抗肿瘤作用。对无胸腺的Balb/C小鼠进行亚致死剂量照射，然后移植人Hep3B肝癌细胞。从具有免疫活性的Balb/C小鼠制备的NKT细胞在体外与肝癌衍生抗原（A组）、Hep3B细胞（B组）或牛血清白蛋白（BSA，C组）一起孵育，然后过继转移到荷肝癌小鼠体内（每只小鼠1×10⁶个NKT细胞）。D组小鼠未进行NKT细胞移植。E组小鼠移植了来自乙肝免疫供体的1×10⁶个NKT细胞。对小鼠的肿瘤大小和重量进行跟踪。为了确定抗肿瘤作用的机制，通过流式细胞术分析脾内淋巴细胞群体中的NKT、CD4⁺和CD8⁺淋巴细胞亚群；通过蛋白质免疫印迹法评估脾细胞中信号转导和转录激活因子1、 STAT4和STAT6的表达，并通过酶联免疫吸附测定法测量血清细胞因子水平。用肝癌衍生抗原脉冲处理的NKT细胞（A组）和来自免疫供体的NKT细胞（E组）的过继转移导致肿瘤在4周内完全消失，并减轻体重减轻（A组和E组分别减轻6.5%和7%）。相比之下，B组、C组和D组的小鼠出现大的坏死肿瘤和严重体重减轻（B组、C组和D组体重减轻分别为21%、17%和23%）。A组和E组的NKT/CD4和CD8/CD4比值显著增加（NKT/CD4比值：A组和E组分别为12.3和17.6，而B组、C组和D组分别为6.4、4.8和5.6；CD8/CD4比值：A组和E组分别为41和19.8，而B组、C组和D组分别为6.5、11.8和3.2）。转录因子STAT4的表达在A组明显，但在B - D组中未观察到。A组和E组的血清干扰素γ、白细胞介素12和白细胞介素4水平升高。用负载在树突状细胞上的肝癌衍生抗原体外处理的NKT淋巴细胞和来自免疫供体的NKT细胞的过继转移导致小鼠肝癌受到抑制。NKT介导的抗肿瘤活性与NKT和CD8⁺T淋巴细胞数量增加、STAT4表达增加（IL - 12活性的标志物）以及促炎细胞因子干扰素γ、白细胞介素12和白细胞介素4的血清水平升高有关。NKT淋巴细胞的体外调节有望成为一种新的肝癌免疫治疗模式。
